The major goal of this project is the investigation of the molecular mechanisms of mutations arising at the hypoxanthine-guanine phosphoribosyltransferase (HPRT; E.C.2.4.2.8) gene locus in man which result in Lesch-Nyhan syndrome or gouty arthritis. This analysis will first require the molecular cloning of the complementary DNA (cDNA) sequence derived from messenger RNA coding for the normal human HPRT enzyme. This will be accomplished by standard techniques for recombinant plasmit constructions, relying on the observed homology with mouse HPRT cDNA sequences for identification of the recombinants carrying human HPRT cDNA inserts. After isolation of full-length cDNA recombinants, the complete nucleotide sequence will be established. Plasmids with cDNA inserts will also be used to isolate genomic recombinants from human DNA libraries constructed in bacteriophage lambda for subsequent determination of the overall gene structure of this locus. Specific DNA fragments from this gene will then be used as probes to classify naturally-occurring mutations oat this locus by DNA and RNA hybridization techniques in cell lines derived from patients with Lesch-Nyhan disease. Subsequent functional and sequence analyses will be used to identify the mutational mechanisms at the molecular level. In addition, cloned sequences will be used to identify restriction fragment length polymorphisms at this locus and explore their possible linkage to other related X-linked heritable diseases.